Sphingolipids are recognized as signaling mediators in a growing number of pathways, and represent potential targets to address many diseases. The study of sphingolipid signaling in yeast has created a number of breakthroughs in the field, and has the potential to lead future advances. The aim of this article is to provide an inclusive view of two major frontiers in yeast sphingolipid signaling. In the first section, several key studies in the field of sphingolipidomics are consolidated to create a yeast sphingolipidome that ranks nearly all known sphingolipid species by their level in a resting yeast cell. The second section presents an overview of most known phenotypes identified for sphingolipid gene mutants, presented with the intention of illuminating not yet discovered connections outside and inside of the field.
The lipid membrane not only provides a rich interface with an array of receptor signaling complexes with which a cell communicates, but it also serves as a source of lipid derived bioactive molecules. In pathologic conditions of acute lung injury (ALI) associated with activation of oxidative stress, unsaturated phosphatidyl cholines overlooking a luminal space undergo oxidation leading to generation of fragmented phospholipids such as 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (lysoPC), or 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) full length oxygenation products (oxPAPC). Using Langmuir monolayers as models of the lipid bilayer, we evaluated the propensity of these phospholipids to solubilize from the cell membrane. The results suggest that lysoPC is rapidly released as it is produced, while oxPAPC has a longer membrane bound lifetime. After being released from cell membranes, these oxidized phospholipids exhibit potent agonist-like effects on neighboring cells. Therefore, we correlate the presence of the two phospholipid groups with the onset and resolution of increased vascular leakiness associated with ALI through testing their effect on vascular endothelial barrier integrity. Our work shows that cells respond differently to these two groups of products of phosphatidyl choline oxidation. LysoPC disrupts cell–cell junctions and increases endothelial permeability while oxPAPC enhances endothelial barrier. These data suggest a model whereby rapid release of lysoPC results in onset of ALI associated vascular leak, and the release of a reserve of oxPAPC as oxidative stress subsides restores the vascular barrier properties.
Oxidized phospholipid; Cell mechanics; Langmuir monolayer; Surface thermodynamics; Gibbs isotherm
The first total synthesis for the novel fatty acid (±)-2-methoxy-6-icosynoic acid was accomplished in seven steps and in a 14% overall yield starting from 2-(4-bromobutoxy)- tetrahydro-2H-pyran. The title compound displayed an EC50 = 23 ± 1 µM against the human SHSY5Y neuroblastoma cell line and an EC50 = 26 ± 1 µM against the human adenocarcinoma cervix cell line (HeLa) after 48 h of exposure. The corresponding non-methoxylated analog 6- icosynoic acid did not display cytotoxicity (EC50 > 500 µM) towards the studied cell lines as well as the 2-methoxyicosanoic acid (EC50 > 300 µM). The critical micelle concentration (CMC = 20–30 µM) for the (±)-2-methoxy-6-icosynoic acid was also determined. It was found that α- methoxylation decreases the CMC of a fatty acid.
Cancer; Neuroblastoma; Methoxylated Fatty Acids; Micelles; Synthesis
The 2-alkynoic fatty acids are an interesting group of synthetic compounds that display antimycobacterial, antifungal, anticancer, and pesticidal activities but their antiprotozoal activity has received little attention until recently. In this review we have summarized our present knowledge of the biomedical potential of the 2-hexadecynoic acid (2-HDA) and 2-octadecynoic acid (2-ODA) together with several mechanistic pieces of work attesting to the fact that these compounds, and their metabolites, are good fatty acid biosynthesis inhibitors. The antiprotozoal activity of 2-HDA and 2-ODA against Leishmania donovani and Plasmodium falciparum, parasites responsible for visceral leishmaniasis and malaria, respectively, is also reviewed. The evidence obtained so far supports the fact that these fatty acids are good inhibitors of the L. donovani DNA topoisomerase IB enzyme (LdTopIB) and the potency of LdTopIB inhibition is chain length dependent. We also demonstrate the generality of the antiprotozoal activity of 2-HDA and 2-ODA against P. falciparum, and review our present knowledge of their inhibition of key P. falciparum enzymes such as PfFabZ, PfFabG, and PfFabI together with some possible modes of inhibition. Recent research by our group has also demonstrated that 2-ODA displays antineoplastic activity, specifically against the neuroblastoma SH-SY5Y cell line via lactate dehydrogenase (LDH) release, which is a cell death mechanism principally associated to necrosis. This is the first comprehensive review of the medicinal chemistry of this interesting group of acetylenic fatty acids.
2-Alkynoic fatty acids; Leishmaniasis; Leishmania donovani; Malaria; Necrosis; Neuroblastoma; Plasmodium falciparum; Topoisomerase IB
Peripheral membrane proteins bound to lipids on bilayer surfaces play central roles in a wide array of cellular processes, including many signaling pathways. These proteins diffuse in the plane of the bilayer and often undergo complex reactions involving the binding of regulatory and substrate lipids and proteins they encounter during their 2-D diffusion. Some peripheral proteins, for example pleckstrin homology (PH) domains, dock to the bilayer in a relatively shallow position with little penetration into the bilayer. Other peripheral proteins exhibit more complex bilayer contacts, for example classical protein kinase C isoforms (PKCs) bind as many as six lipids in stepwise fashion, resulting in the penetration of three PKC domains (C1A, C1B, C2) into the bilayer headgroup and hydrocarbon regions. A molecular understanding of the molecular features that control the diffusion speeds of proteins bound to supported bilayers would enable key molecular information to be extracted from experimental diffusion constants, revealing protein-lipid and protein-bilayer interactions difficult to study by other methods. The present study investigates a range of 11 different peripheral protein constructs comprised by 1 to 3 distinct domains (PH, C1A, C1B, C2, anti-lipid antibody). By combining these constructs with various combinations of target lipids, the study measures 2-D diffusion constants on supported bilayers for 17 different protein-lipid complexes. The resulting experimental diffusion constants, together with the known membrane interaction parameters of each complex, are used to analyze the molecular features correlated with diffusional slowing and bilayer friction. The findings show that both 1) individual bound lipids and 2) individual protein domains that penetrate into the hydrocarbon core make additive contributions to the friction against the bilayer, thereby defining the 2-D diffusion constant. An empirical formula is developed that accurately estimates the diffusion constant and bilayer friction of a peripheral protein in terms of its number of bound lipids and its geometry of penetration into the bilayer hydrocarbon core, yielding an excellent global best fit (R2 of 0.97) to the experimental diffusion constants. Finally, the observed additivity of the frictional contributions suggests that further development of current theory describing bilayer dynamics may be needed. The present findings provide constraints that will be useful in such theory development.
PKC; GRP1; cPLA2; PH domain; C1 domain; C2 domain; phosphatidylinositol lipid
Phospholipase A2 (PLA2) enzymes catalyze the hydrolysis of the sn-2 ester linkage of glycerophospholipids to produce fatty acids and lysophospholipids. A significant number of mammalian phospholipases comprise a family of secreted PLA2 enzymes, found in specific tissues and cellular locations, exhibiting unique enzymatic properties and distinct biological functions. Development of new real-time spectrofluorimetric PLA2 assays should facilitate the kinetic characterization and mechanistic elucidation of the isozymes in vitro, with the potential applicability to detect and measure catalytic PLA2 activity in tissues and cellular locations. Here we report a new synthesis of double-labeled phosphatidylcholine analogues with chain-terminal reporter groups including coumarin fluorophores for fluorescence resonance energy transfer (FRET)-based kinetic studies of PLA2 enzymes. The use of coumarin derivatives as fluorescent labels provides reporter groups with substantially decreased size compared to the first generation of donor-acceptor pairs of fluorescent phospholipids. The key advantage of the design is to interfere less with the physicochemical properties of the acyl chains, thereby improving the substrate quality of the synthetic probes. In order to assess the impact of the fluorophore substituents on the catalytic hydrolysis and on the phospholipid packing in the lipid-water interface of the assay, we used the experimentally determined specific activity of bee-venom phospholipase A2 as a model for the secretory PLA2 enzymes. Specifically, the rate of PLA2 hydrolysis of the coumarin labeled phosphatidylcholine analogues was less than three times slower than natural substrate dipalmitoyl phosphatidylcholine (DPPC) under the same experimental conditions. Furthermore, variation of the mole fraction of the synthetic phosphatidylcholine vs. that of the natural DPPC substrate showed nearly ideal mixing behavior in the phospholipid-surfactant aggregates of the assay. The synthesis provides a rapid and efficient method for preparation of new synthetic phosphatidylcholines with the desired target structures for enzymatic and physicochemical studies.
Class A G-protein coupled receptors (GPCRs) are thought to have a common topology that includes seven transmembrane alpha helices (TMHs) that are arranged to form a closed bundle. This bundle forms the ligand binding pocket into which ligands are commonly thought to enter via the extracellular milieu. This ligand approach direction makes sense for GPCRs that have small positively charged ligands, such as the beta-2-adrenergic or the dopamine D2 receptor. However, there is a growing sub-group of Class A GPCRs that bind lipid-derived endogenous ligands, such as the cannabinoid CB1 and CB2 receptors (with endogenous ligands, N-arachidonoylethanolamine (anandamide) and sn-2-arachidonylglycerol (2-AG)) and the S1P1-5 receptors (with endogenous ligand, sphingosine-1-phosphate). Even the widely studied Class A GPCR, rhodopsin, binds a highly lipophillic chromophore (11-cis-retinal). For these receptors, ligand approach from the extracellular milieu has seemed unlikely given that the ligands of these receptors readily partition into lipid or are actually synthesized in the lipid bilayer. The recent X-ray-crystal structure of the sub-type 1 sphingosine-1-phosphate receptor (S1P1) provides important information on the key structural variations that may be the hallmarks for a Class A GPCR that binds lipid-derived ligands. These include an extracellular domain that is closed off to the extracellular milieu and the existence of an opening between transmembrane helices that may serve as a portal for ligand entry via the lipid bilayer. This review examines structural aspects that the cannabinoid receptors may share with the S1P1 receptor based upon sequence homology. This review also examines experimental and simulation results that suggest ligand entry via a lipid portal is quite likely for this emerging sub-group.
Cannabinoid; Sphingosine-1-phosphate; GPCR; Crystal structure; Lipid portal
Meibomian gland secretions (or meibum) are produced by holocrine meibomian glands and are secreted in melted form onto the ocular surface of humans and animals to form a protective tear film lipid layer (TFLL). Its protective effect strongly depends on the composition and, hence, thermotropic behavior of meibum. The goal of our study was to quantitatively evaluate the melting characteristics of human meibum and model lipid mixtures using differential scanning microcalorimetry. Standard calorimetric parameters, e.g. changes in calorimetric enthalpy, transition temperatures T(m), cooperativity of melting etc. were assessed. We found that thermotropic behavior of meibum resembled that of relatively simple mixtures of unsaturated wax esters, but showed a lower change in calorimetric enthalpy, which can be indicative of a looser packing of lipids in meibum compared with pure standards and their simple mixtures. The cooperativity of melting of meibomian lipids was comparable to that of an equimolar mixture of four oleic-acid based wax esters. We demonstrated that the phase transitions in meibum start at about 10 to 15 °C and end at 35-36 °C, with T(m) being about 30 °C. The highly asymmetrical shape of the thermotropic peak of meibum is important for the physiology and biophysics of TFLL.
cooperativity; meibum; meibomian gland secretions; melting; phase transitions; microcalorimetry; lipids; wax esters
Minimally-invasive image-guided tumor ablation using short duration heating via needle-like applicators using energies such as radiofrequency or microwave has seen increasing clinical use to treat focal liver, renal, breast, bone, and lung tumors. Potential benefits of this thermal therapy include reduced morbidity and mortality compared to standard surgical resection and ability to treat non-surgical patients. However, improvements to this technique are required as achieving complete ablation in many cases can be challenging particularly at margins of tumors > 3 cm in diameter and adjacent to blood vessels. Thus, one very promising strategy has been to combine thermal tumor ablation with adjuvant nanoparticle-based chemotherapy agents to improve efficiency. Here, we will primarily review principles of thermal ablation to provide a framework for understanding the mechanisms of combination therapy, and review the studies on combination therapy, including presenting preliminary data on the role of such variables as nanoparticle size and thermal dose on improving combination therapy outcome. We will discuss how thermal ablation can also be used to improve overall intratumoral drug accumulation and nanoparticle content release. Finally, in this article we will further describe the appealing off-shoot approach of utilizing thermal ablation techniques not as the primary treatment, but rather, as a means to improve efficiency of intratumoral nanoparticle drug delivery.
Serving as a crucial component of mammalian cells, cholesterol critically regulates the functions of biomembranes. This review focuses on a specific property of cholesterol and other sterols: the tilt modulus χ that quantifies the energetic cost of tilting sterol molecules inside the lipid membrane. We show how χ is involved in determining properties of cholesterol-containing membranes, and detail a novel approach to quantify its value from atomistic molecular dynamics (MD) simulations. Specifically, we link χ with other structural, thermodynamic, and mechanical properties of cholesterol-containing lipid membranes, and delineate how this useful parameter can be obtained from the sterol tilt probability distributions derived from relatively small-scale unbiased MD simulations. We demonstrate how the tilt modulus quantitatively describes the aligning field that sterol molecules create inside the phospholipid bilayers, and we relate χ to the bending rigidity of the lipid bilayer through effective tilt and splay energy contributions to the elastic deformations. Moreover, we show how χ can conveniently characterize the “condensing effect” of cholesterol on phospholipids. Finally, we demonstrate the importance of this cholesterol aligning field to the proper folding and interactions of membrane peptides. Given the relative ease of obtaining the tilt modulus from atomistic simulations, we propose that χ can be routinely used to characterize the mechanical properties of sterol/lipid bilayers, and can also serve as a required fitting parameter in multi-scaled simulations of lipid membrane models to relate the different levels of coarse-grained details.
cholesterol orientation; lipid tilt modulus; transitions in liquid crystals; 7DHC; SLOS; dynorphin; molecular dynamics simulations
Cellular lipid membranes are spatially inhomogeneous soft materials. Materials properties such as pressure and surface tension thus show important microscopic-scale variation that is critical to many biological functions. We present a means to calculate pressure and surface tension in a 3D-resolved manner within molecular-dynamics simulations and show how such measurements can yield important insight. We also present the first corrections to local virial and pressure fields to account for the constraints typically used in lipid simulations that otherwise cause problems in highly oriented systems such as bilayers. Based on simulations of an asymmetric bacterial ion channel in a POPC bilayer, we demonstrate how 3D-resolved pressure can probe for both short-range and long-range effects from the protein on the membrane environment. We also show how surface tension is a sensitive metric for inter-leaflet equilibrium and can be used to detect even subtle imbalances between bilayer leaflets in a membrane-protein simulation. Since surface tension is known to modulate the function of many proteins, this effect is an important consideration for predictions of ion channel function. We outline a strategy by which our local pressure measurements, which we make available within a version of the GROMACS simulation package, may be used to design optimally equilibrated membrane-protein simulations.
Lipid bilayer; Molecular dynamics; Lateral pressure; Ion channel
Many observations of the role of the membrane in the function and organization of transmembrane (TM) proteins have been explained in terms of hydrophobic mismatch between the membrane and the inserted protein. For a quantitative investigation of this mechanism in the lipid-protein interactions of functionally relevant conformations adopted by a multi-TM segment protein, the bacterial Leucine Transporter (LeuT), we employed a novel method, Continuum-Molecular Dynamics (CTMD), that quantifies the energetics of hydrophobic mismatch by combining the elastic continuum theory of membrane deformations with an atomistic level description of the radially asymmetric membrane-protein interface from MD simulations. LeuT has been serving as a model for structure-function studies of the mammalian Neurotransmitter:Sodium Symporters (NSSs), such as the dopamine and serotonin transporters, which are the subject of intense research in the field of neurotransmission. The membrane models in which LeuT was embedded for these studies were composed of 1-palmitoyl-2-oleoyl-sn -glycero-3-phosphocholine (POPC) lipid, or 3:1 mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) lipids. The results show that deformation of the host membrane alone is not sufficient to alleviate the hydrophobic mismatch at specific residues of LeuT. The calculations reveal significant membrane thinning and water penetration due to the specific local polar environment produced by the charged K288 of TM7 in LeuT, that is membrane-facing deep inside the hydrophobic milieu of the membrane. This significant perturbation is shown to result in unfavorable polar-hydrophobic interactions at neighboring hydrophobic residues in TM1a and TM7. We show that all the effects attributed to the K288 residue (membrane thinning, water penetration, and the unfavorable polar-hydrophobic interactions at TM1a and TM7), are abolished in calculations with the K288A mutant. The involvement of hydrophobic mismatch is somewhat different in the functionally distinct conformations (outward-open, occluded, inward-open) of LeuT, and the differences are shown to connect to structural elements (e.g., TM1a) known to play key roles in transport. This finding suggests a mechanistic hypothesis for the enhanced transport activity observed for the K288A mutant, suggesting that the unfavorable hydrophobic-hydrophilic interactions hinder the motion of TM1a in the functionally-relevant conformational transition to the inward-open state. Various extents of such unfavorable interactions, involving exposure to the lipid environment of adjacent hydrophobic and polar residues, are common in multi-segment transmembrane proteins, and must be considered to affect functionally relevant conformational transitions.
Lipid-Protein Interactions; Hydrophobic Mismatch; Residual Exposure energy; Leucine Transporter; Neurotransmitter:Sodium Symporters; Multi-segment Transmembrane Proteins; Molecular dynamics simulations; Membrane deformation energy
We present nearly ten microseconds of all-atom simulation data of a G-protein coupled receptor, the human A2A adenosine receptor, bound to four different ligands. Our focus is on binding of cholesterol to the “cholesterol consensus motif,” a cluster of five amino acids on the second and fourth transmembrane helices, which interact with two cholesterols in the intracellular leaflet of the bilayer. We find evidence for a ligand-specific interaction between the CCM and cholesterol, mediated by the rotameric dynamics and configuration of Trp129. Binding of the synthetic agonist UK432097 disrupts hydrogen bonding between Trp129 and Ser47, which activates the rotameric dynamics of Trp129 and disrupts the interaction with one of the two cholesterols. We also investigate the effect of four thermostabilizing mutations, three of which are located on helix two. The conformational stability of helix two has been proposed to be sensitive to interaction with cholesterol in the CCM, suggesting a mechanism for the thermostabilization. However, our data are instead suggestive of a force-field dependent “straightening” of helix two, and therefore offer no basis for rationalizing the effect of the quadruple mutant.
G-protein coupled receptor; Cholesterol; Molecular dynamics
For four decades, since W. Helfrich’s pioneering study of smectic A liquid crystals in 1973, continuum elastic models (CEMs) have been employed as tools to understand the energetics of protein-induced lipid bilayer deformations. Among the assumptions underlying this use is that all relevant protein–lipid interactions can be included in the continuum representation of the protein–bilayer interactions through the physical parameters determined for protein-free bilayers and the choice of boundary conditions at the protein/bilayer interface. To better understand this assumption, we review the general structure of CEMs, examine how different choices of boundary conditions and physical moduli profiles alter the predicted bilayer thickness profiles around gramicidin A (gA) and mitochondrial voltage-dependent anion channels (VDAC), respectively, and compare these profiles with those obtained from all-atom molecular dynamics simulations. We find that the profiles differ qualitatively in the first lipid shell around the channels, indicating that the CEMs do not capture accurately the consequences of the protein-induced local changes in lipid bilayer dynamics. Therefore, one needs to be careful when interpreting the results of CEM-based analyses of lipid bilayer-membrane protein interactions.
Gramicidin A (gA); Voltage dependent anion channel (VDAC); Molecular dynamics simulations; Protein–lipid interactions
We characterize the allylic epoxyalcohols and their trihydroxy hydrolysis products generated from 9R- and 9S-hydroperoxy-octadecenoic acid (HPODE) under non-enzymatic conditions, reaction with hematin and subsequent acid hydrolysis, and enzymatic conditions, incubation with Beta vulgaris containing a hydroperoxide isomerase and epoxide hydrolase. The products were resolved by HPLC and the regio and stereo-chemistry of the transformations were determined through a combination of 1H NMR and GC-MS analysis of dimethoxypropane derivatives. Four trihydroxy isomers were identified upon mild acid hydrolysis of 9S,10S-trans-epoxy-11E-13S-hydroxyoctadecenoate: 9S,10R,13S, 9S,12R,13S, 9S,10S,13S and 9S,12S,13S-trihydroxy-octadecenoic acids, in the ratio 40:26:22:12. We also identified a prominent -ketol rearrangement product from the hydrolysis as mainly the 9-hydroxy-10E-13-oxo isomer. Short incubation (5 min) of 9R- and 9S-HPODE with Beta vulgaris extract yielded the 9R- and 9S-hydroxy-10E-12R,13S-cis-epoxy products respectively. Longer incubation (60 min) gave one specific hydrolysis product via epoxide hydrolase, the 9R/S,12S,13S-trihydroxyoctadecenoate. These studies provide a practical approach for the isolation and characterization of allylic epoxy alcohol and trihydroxy products using a combination of HPLC, GC-MS and 1H NMR.
9-HPODE; trihydroxyoctadecenoic acid; linoleic acid; epoxyalcohol synthase; epoxide hydrolase; hematin
When giant unilamellar vesicles (GUV) composed of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, and cholesterol are treated with PlcHR2, a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa, the initial stages of lipid hydrolysis do not cause large changes in vesicle morphology (Ibarguren et al., 2011). However, when hydrolysis progresses confocal fluorescence microscopy reveals the formation of lipid aggregates, whose morphology is not compatible with that of bilayers. Smaller vesicles or droplets can also be seen inside the GUV. Our studies indicate that these aggregates or droplets are enriched in the non-lamellar lipid ceramide, an end-product of PlcHR2 reaction. Moreover, the aggregates/droplets appear enriched in the hydrolytic enzyme PlcHR2. At a final stage GUVs containing the enzyme-enriched droplets disintegrate and vanish from the microscope field. The observed non-lamellar enzyme-rich structures may be related to intermediates in the process of aggregation and fusion although the experimental design prevents vesicle free diffusion in the aqueous medium, thus actual aggregation or fusion cannot be observed.
Phospholipase C/sphingomyelinase HR2; Pseudomonas aeruginosa; non-lamellar phases; lipid aggregates
Three novel polymerizable amphiphiles with a sorbyl-substituted head group were synthesized and systematically characterized. These amphiphiles are neutral in charge. None of these molecules forms vesicles by itself, presumably due to lack of amphiphilicity and/or extensive head group interaction. Therefore, mixed vesicles were formed with other fluid lipids such as DPenPC, eggPC, or DOPC. We investigated the properties of these mixtures in both vesicles and Langmuir films. The Langmuir isotherms show formation of monolayers by all three molecules. However, the isotherms for mixed monolayers suggest that two components are largely immiscible to the mixing lipid. Under polymerization conditions, mixed vesicles of these amphiphiles form oligomers, suggesting that in spite of a larger head group, they form mesophases.
Sorbyl groups; polymerizable lipids; Langmuir isotherms; mixed liposomes
Sphingadienes are chemopreventive agents that act by blocking signaling pathways that are activated in cancer. A practical synthesis of 4,6- and 4,8-sphingadienes on a scale of gram quantities is reported here in order to allow evaluation of the biological properties of these sphingolipids. The key steps in the preparation of 4,6-sphingadiene (1a) are an intramolecular cyclization of N-Boc derivative 5a to oxazolidinone derivative 6a, followed by conversion to carbamate intermediate 7a and base-mediated hydrolysis to afford the product without further purification. 4,8-Sphingadiene (1b) was prepared in a similar fashion; the requisite trans-γ,δ-unsaturated aldehyde 15 was prepared by an ester enolate Ireland-Claisen rearrangement.
Lipid synthesis; Sphingadienine; Sphingolipid; Sphingoid base
Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has emerged as a novel powerful MS methodology that has the ability to generate both molecular and spatial information within a tissue section. Application of this technology as a new type of biochemical lipid microscopy may lead to new discoveries of the lipid metabolism and biomarkers associated with area-specific alterations or damage under stress/disease conditions such as traumatic brain injury or acute lung injury, among others. However there are limitations in the range of what it can detect as compared with liquid chromatography-MS (LC-MS) of a lipid extract from a tissue section. The goal of the current work was to critically consider remarkable new opportunities along with the limitations and approaches for further improvements of MALDI-MSI. Based on our experimental data and assessments, improvements of the spectral and spatial resolution, sensitivity and specificity towards low abundance species of lipids are proposed. This is followed by a review of the current literature, including methodologies that other laboratories have used to overcome these challenges.
Lipidomics; MALDI imaging; Traumatic brain injury; Acute lung injury; Lipid peroxidation; Docosahexaenoic acid
An analysis of electron spin resonance (ESR) spectra from compositions along the liquid-ordered (Lo) and liquid-disordered (Ld) coexistence curve from the brain-sphingomyelin/dioleoylphosphatidylcholine/cholesterol (SPM/DOPC/Chol) model lipid system was performed to characterize the dynamic structure on a molecular level of these coexisting phases. We obtained 200 continuous-wave ESR spectra from glycerophospholipid spin-labels labeled at the 5, 7, 10, 12, 14, and 16 carbon positions of the 2nd acyl chain, a sphingomyelin spin-label labeled at the 14 carbon position of the amide-linked acyl chain, a headgroup-labeled glycerophospholipid, a headgroup-labeled sphingomyelin, and the cholesterol analogue spin-label cholestane all within multi-lamellar vesicle suspensions at room temperature. The spectra were analyzed using the MOMD (microscopic-order macroscopic-disorder) model to provide the rotational diffusion rates and order parameters which characterize the local molecular dynamics in these phases. The analysis also incorporated the known critical point and invariant points of the neighboring three-phase triangle along the coexistence curve. The variation in the molecular dynamic structures of coexisting Lo and Ld compositions as one moves toward the critical point is discussed. Based on these results, a molecular model of the Lo phase is proposed incorporating the “condensing effect” of cholesterol on the phospholipid acyl chain dynamics and ordering and the “umbrella model” of the phospholipid headgroup dynamics and ordering.
liquid-ordered phase; coexisting phases; bilayer structure; umbrella model; condensing effect; critical point
Signaling lipids control many of the most important biological pathways, typically by recruiting cognate protein binding targets to cell surfaces, thereby regulating both their function and subcellular localization. A critical family of signaling lipids is that of the phosphatidylinositol polyphosphates (PIPns), which is composed of seven isomers that vary based on phosphorylation pattern. A key protein that is activated upon PIPn binding is Akt, which then plays important roles in regulating the cell cycle, and is thus aberrant in disease. Characterization of protein–PIPn binding interactions is hindered by the complexity of the membrane environment and of the PIPn structures. Herein, we describe two rapid assays of use for characterizing protein–PIPn binding interactions. First, a microplate-based binding assay was devised to characterize the binding of effectors to immobilized synthetic PIPn headgroup–biotin conjugates corresponding to all seven isomers. The assay was implemented for simultaneous analysis of Akt-PH domain, indicating PI(3,4,5)P3 and PI(3,4)P2 as the primary ligands. In addition, density-dependant studies indicated that the amount of ligand immobilized on the surface affected the amplitude of protein binding, but not the affinity, for Akt-PH. Since the PIPn ligand motifs used in this analysis lack the membrane environment and glycerolipid backbone, yet still exhibit high-affinity protein binding, these results narrow down the structural requirements for Akt recognition. Additionally, binding detection was also achieved through microarray analysis via the robotic pin printing of ligands onto glass slides in a miniaturized format. Here, fluorescence-based detection provided sensitive detection of binding using minimal amounts of materials. Due to their high-throughput and versatile attributes, these assays provide invaluable tools for probing and perturbing protein–membrane binding interactions.
Phospholipids; membranes; phosphoinositides; cell surface; microarray
We synthesized and characterized a series of zwitterionic, acetate-terminated, quaternized amine diacyl lipids (AQ). These lipids have an inverted headgroup orientation as compared to naturally occurring phosphatidylcholine (PC) lipids; the cationic group is anchored at the membrane interface, while the anionic group extends into the aqueous phase. AQ lipids preferentially interact with highly polarizable anions (ClO4−) over less polarizable ions (Cl−), in accord with the Hofmeister series, as measured by the change in zeta potential of AQ liposomes. Conversely, AQ lipids have a weaker association with calcium than do PC lipids. The transition temperatures (Tm) of the AQ lipids are similar to the Tm observed with phosphatidylethanolamine (PE) lipids of the same chain length. AQ lipids form large lipid sheets after heating and sonication; however, in the presence of cholesterol, (Chol) these lipids form stable liposomes that encapsulate carboxyfluorescein. The AQ:Chol liposomes retain their contents in the presence of serum at 37 °C, and when injected intravenously into mice, their organ biodistribution is similar to that observed with PC:Chol liposomes. AQ lipids demonstrate that modulating the headgroup charge orientation significantly alters the biophysical properties of liposomes. For the drug carrier field, these new materials provide a non-phosphate containing zwitterlipid for the production of lipid vesicles.
charge orientation; drug carriers; lipid vesicle; nanotechnology; permeability
Tetraazamacrocyclic complexes of transition metals provide useful units for incorporating multiple coordination interactions into a single protein binding molecule. They can be designed with available sites for protein interactions with donor atom containing amino acid side chains or have labile ligands such as H2O allowing facile exchange. Three configurationally restricted nickel(II) cyclam complexes with either one or two macrocyclic rings were synthesised and their ability to abrogate the CXCR4 chemokine receptor signalling process was assessed (IC50 = 8320, 194 and 14 nM). Analogues were characterised crystallographically to determine the geometric parameters of acetate binding as a model for aspartate. The most active nickel(II) compound was tested in several anti-HIV assays against representative viral strains showing highly potent EC50 values down to 13 nM against CXCR4 using viruses with no observed cellular cytotoxicity (CC50 > 125 μM).